Storage and Stability:

TRIsure™ TRIsure is shipped at room temperature. For optimal performance, we recommend to store at +4 °C.
Expiry:
Catalog Numbers When stored under the recommended conditions and handled correctly, full activity is retained until the expiry
TRIsure date on the outer box label.
BIO-38032 100 mL
Safety Precautions:
TRIsure BIO-38033 200 mL Toxic in contact with skin. Toxic if swallowed. Causes burns. Please refer to the material safety data
sheet for information regarding hazards and safe handling practice.
Batch No.: See vial

Store at +4 oC

Signal word : DANGER

Notes:
For research or further manufacturing use only.
Trademarks:
TRIsure, MyTaq and SensiFAST are trademarks of Bioline Reagents Ltd.

Features Applications
• Quick isolation of high-quality total RNA, DNA and/or protein from a • Purified RNA is ideal for any downstream application such as
single sample RT-PCR, in vitro translation, northern blotting, RNase protection
• Performs well with large or small amounts of tissue or cells assays or dot blot hybridization
• Ready-to-use solution • Purified DNA can be used for PCR and Southern blotting
• Purified protein can be used for western blotting

Description
TRIsure™ is a complete, ready-to-use reagent for the isolation of RNA, DNA and protein from cells and tissues. Using TRIsure, a biological sample is
homogenized and lysed before being separated into three phases: an aqueous phase (upper), an organic phase (lower) and an interphase. The RNA is
extracted from the aqueous phase by isopropyl alcohol precipitation. The highly effective RNase inhibitory property of TRIsure protects the integrity of the
RNA during the lysis and results in the isolation of high-quality material. DNA is precipitated from the organic layer with ethanol. Protein is sequentially
precipitated from the phenol-ethanol supernatant by isopropyl alcohol precipitation.
1 mL of TRIsure is sufficient to isolate RNA and DNA from 1 x 107 cells or 100 mg of tissue. The table below presents typical yields of RNA and DNA from
various starting materials.

Starting material Quantity RNA DNA

Mouse liver 1 mg 2-5 µg 3-4 µg

Mouse kidney 1 mg 5-10 µg 3-4 µg

6
Epithelial cells 1 x 10 cells 8-15 µg 5-7 µg

Fibroblast cells 1 x 106 cells 20-25 µg 5-7 µg

Protocol for the isolation of RNA using TRIsure 3. RNA Precipitation

Transfer the aqueous phase very carefully, without disturbing the interphase
Reagents required (not supplied): to another tube. Precipitate the RNA by mixing with cold isopropyl alcohol.
• Chloroform Use 0.5 mL of isopropyl alcohol per 1 mL of TRIsure used. Incubate samples
for 10 minutes at room temperature then centrifuge at 12,000 x g for 10
• Isopropyl alcohol (chilled)
minutes (or 2600 x g for 30 minutes) at 4 °C.
• 75% ethanol (in DEPC-treated water)
Note: For small quantities of cells, RNase-free Co-precipitant Pink (BIO-37075) can be
• DEPC-treated water or PCR water added to the aqueous phase before addition of isopropyl alcohol to aid visualizing RNA
precipitation. Add 5-10 g of Co-precipitant per 800 L of TRIsure.
1. Homogenization
Tissue: 4. RNA Wash
Homogenize tissue samples in 1 mL of TRIsure per 50-100 mg of tissue. Remove the supernatant. Wash the pellet once with 75% ethanol, adding at
For small quantities of tissue (1-10 mg), add 800 µL of TRIsure. For least 1 mL of ethanol per 1 mL of TRIsure used. Vortex samples and
samples of fat tissue, a layer of fat may accumulate at the top, which centrifuge at 7500 x g for 5 minutes at 4 °C.
should be removed. Note: At this stage, samples can be stored for one week at 4 °C, or 12 months at
Plant tissue -20 °C.
Following homogenization, insoluble material is removed by centrifugation 5. Re-dissolving the RNA
at 12,000 x g for 10 minutes at 4 °C. Transfer the cleared homogenate to Air-dry the pellet and dissolve in PCR water or DEPC-treated water by pipet-
a fresh tube. ting the solution up and down. Incubate for 10 minutes at 60 °C if necessary.
Cells Grown on Monolayer: Store RNA at -70 °C.
Lyse cells directly in a culture dish or flask by adding 1 mL of TRIsure per
10cm2 growth area, pipette the cell lysate several times to ensure Protocol for the isolation of DNA using TRIsure
sufficient cell disruption. Reagents required (not supplied):
Cells Grown in Suspension: • 100% ethanol
Pellet cells at 200 x g for 5 minutes at room temperature. Lyse cells with • 0.1 M sodium citrate in 10% ethanol
1 mL of TRIsure per 5 x 106 cells and pass the lysate several times
through a pipette tip. For small quantities of cells (102 -106), lyse cells in • 75% ethanol
800 µL of TRIsure. • 8 mM NaOH
Note: At this stage, samples can be stored for at least one month at -70 °C. • DEPC-treated water or PCR water
2. Phase Separation After homogenization and phase separation the upper aqueous phase is
Incubate samples for 5 minutes at room temperature. Add 0.2 mL of removed for optional RNA precipitation, leaving the interphase and the
chloroform per 1 mL of TRIsure used. Cap tubes securely and shake organic phase for sequential isolation of DNA and protein.
vigorously by hand for 15 seconds.
Note: The interphase and organic phase can be stored overnight at 4 °C.
Incubate samples for 3 minutes at room temperature. Centrifuge samples
at 12,000 x g for 15 minutes (or 2600 x g for 30 minutes) at 4 °C. The 1. DNA Precipitation
sample will separate into a pale green, organic phase, an interphase, and Remove any remaining aqueous phase overlying the interphase (step 3 of
a colorless upper aqueous phase. the RNA isolation protocol). Add 0.3 mL of 100% ethanol per 1 mL of
The aqueous phase is removed for RNA extraction. TRIsure used, and mix samples by inversion. Leave samples at room
temperature for 3 minutes, then centrifuge at 2000 x g for 5 minutes at 4 °C.
Note: At this stage, samples can be stored for at least one month at 4 °C.

PI-50260 V7 Website: www.bioline.com email: info@meridianlifescience.com

2. DNA Wash
Remove the supernatant to waste or retain for protein isolation. Wash the
DNA pellet, with 1 mL of 0.1 M sodium citrate in 10% ethanol per 1 mL of
TRIsure used, and mix for 30 minutes at room temperature. Centrifuge
samples at 2000 x g for 5 minutes at 4 °C. Two washes are usually
sufficient, however for large pellets containing >200 µg of DNA an
additional wash may be necessary.
Following the wash steps, add 1.5 mL of 75% ethanol per 1 mL of TRIsure
used. Mix for 20 minutes at room temperature, then centrifuge samples at
2000 x g for 5 minutes at 4 °C.
3. Re-dissolving the DNA
Air-dry the pellet for 15 minutes. Resuspend the pellet in 8 mM NaOH
Remove any insoluble material by centrifugation at 12000 x g for 10
minutes and then transfer the supernatant to another tube.
Note: Samples dissolved in 8 mM NaOH can be stored overnight at 4 °C. For
long-term storage, adjust the pH to 7.5, and add EDTA to achieve a 1 mM concen-
tration. Store at -20 °C.
Protocol for the isolation of protein using TRIsure
Reagents required (not supplied):
• Isopropyl alcohol
• 0.3 M Guanidine hydrochloride in 95% ethanol
• Ethanol
• 1% SDS
1. Protein Precipitation
To the retained supernatant (step 2 of the DNA isolation protocol) add 1.5 mL
of isopropyl alcohol per 1 mL of TRIsure used. Mix samples for 10 minutes at
room temperature then centrifuge at 12000 x g for 10 minutes at 4 °C.
2. Protein Wash
Remove supernatant and wash the protein pellet twice. To wash the protein
pellet add 2 mL of 0.3 M guanidine hydrochloride in 95% ethanol per 1 mL of
TRIsure used. Mix for 20 minutes at room temperature then centrifuge at
7500 x g for 5 minutes at 4 °C.
Note: At this stage, samples can be stored for at least one month at 4 °C, or 12 months
at -20 °C.
Following the washing steps, add 2 mL of ethanol and vortex. Mix for 20
minutes at room temperature then centrifuge at 7500 x g for 5 minutes at
4 °C.
Note: At this stage, samples can be stored for at least one month at 4 °C, or 12 months
at -20 °C.
3. Re-dissolving the Protein
Vacuum dry the protein pellet for 5-10 minutes. Dissolve in 1% SDS by
pipetting up and down. For difficult samples incubate at 50 °C. Remove any
insoluble material by centrifugation at 10000 x g for 10 minutes at 4 °C and
then transfer the supernatant to another tube. Store protein at -20 °C.
References:
Chomczynski, P. and Sacchi, N. Anal. Biochem. 162: 156-159. (1987)
Chomczynski, P. BioTechniques 15: 532-537. (1993)

Troubleshooting Guide

Problem Possible Cause Recommendation

Interphase/organic phase pipetted

Do not attempt to draw off the entire aqueous layer after phase separation.
up with aqueous phase
DNA contamination or Insufficient removal of the aqueous
RNA contamination Remove remnants of the aqueous phase prior to DNA precipitation.
phase from the organic phase
Insufficient wash of the DNA pellet Make sure pellet is washed with 0.1 M sodium citrate in 10% ethanol.
Insufficient homogenisation or lysis Decrease the amount of starting material. Mince tissues into smaller pieces and make sure it
of samples is completely immersed in TRIsure for optimal lysis.
Insufficient solubilization of RNA,
Low RNA yield Increase the solubilization by pipetting the sample repeatedly, heat the sample to 60 °C.
DNA or protein pellet
If starting sample is small, the pellet may not be easily visualized after precipitation, so care
Loss of pellet
must be taken when removing the supernatant from the pellet.
Samples were not immediately
Sample must be processed or frozen immediately after collection.
processed or frozen after collection
DNA is degraded, Isolated RNA, DNA or protein
DNA is degraded or preparations were stored at the Store RNA samples at -80 °C. Store DNA and protein samples at -20 °C.
protein is degraded incorrect temperature
Protocol must be carried out carefully in a DNA-free, RNase-free environment. Addition of
RNase contamination
RNase Inhibitor to the extracted RNA sample can help prevent degradation of the sample.
Ensure that 1 mL TRIsure per 10 cm 2 area of cells or 5 x 106 cells is used. If problem
Insufficient volume of TRIsure
persists, increase TRIsure volume by 1.5 x.
Low A260/280 for RNA Contamination of interphase layer Pipette off the aqueous phase very carefully. It is important that none of the white interphase
during separation of the RNA- is transferred into your RNA sample, so we recommend that you leave the lower part of the
containing aqueous layer aqueous phase intact.
Phenol was not sufficiently
Low A260/280 for DNA Wash the DNA pellet one additional time in 0.1 M sodium citrate in 10% ethanol.
removed from the DNA preparation

Technical Support Associated Products

If the troubleshooting guide does not solve the difficulty you are Product Name Pack Size Catalog No.
experiencing, please contact Technical Support with details of
the procedure and relevant data. SensiFAST™ cDNA Synthesis Kit 50 Reactions BIO-65053
Email: mbi.tech@meridianlifescience.com
MyTaq™ HS Mix 200 Reactions BIO-25045

SensiFAST™ SYBR® No-ROX

100 Reactions BIO-72001
One-Step Kit

____________________________________________________________________________________________________________________
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UNITED KINGDOM USA GERMANY AUSTRALIA

Tel: +44 (0)20 8830 5300 Tel: +1 901.382.8716 Tel: +49 (0)3371 60222 03 Tel: +61 (0)2 9209 4180
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Website: www.bioline.com email: info@meridianlifescience.com